The proteasome is solely responsible for targeted degradation of specific proteins in eukaryotes, and thus the process of substrate selection and initiation of degradation is the most critical step in regulation of this critical process. A complx of ATPases in the 26S proteasome regulate this critical step, however, it is not known how substrate binding to the proteasome regulates proteasome activity to modulate substrate processing, nor is it known if structural changes in the ATPase's N-domain allow for these regulatory mechanisms. The current working hypothesis is that substrate binding to the extreme N-domain of the ATPase allosterically communicates to the ATPase domains to trigger substrate processing activities. To determine the conformational changes that occur in the N-domain of the proteasomal ATPases, point-specific crosslinks will be used to determine how substrate and nucleotide binding alter the range of N-domain conformational states that exist. Furthermore, to determine the role of the N-domain's conformational changes in proteasome function, conformational changes will be restricted using these established crosslinks and biochemical and biophysical techniques will be employed to assess the functions of these conformational changes. This work is significant because it will define a dynamic mechanism that allows substrates to activate proteasome function, allowing one to understand how the proteasomal ATPases communicate distant messages through the N-domains at a molecular level. This will provide a mechanistic framework to understand how substrate degradation by the proteasome could be perturbed in human diseases. This research is innovative because the tractable in vitro model system has allowed the generation of highly novel preliminary data that offer critical insights into understanding how the N-domains regulate proteasome function.
Virtually every cellular process relies on properly regulated protein degradation, and improper regulation of protein degradation leads to human disease (e.g. cancers, neurodegenerative diseases, cardiomyopathies). The proteasome is solely responsible for targeted protein degradation in eukaryotes, and the process of substrate selection and commitment by the proteasome is the most critical step in proper regulation of protein degradation. The outcomes of the proposed research will have a positive impact because they provide a mechanistic framework to understand how the proteasomal ATPases regulate protein degradation, which will inform the field how this process can be exploited in disease states.
| Snoberger, Aaron; Brettrager, Evan J; Smith, David M (2018) Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing. Nat Commun 9:2374 |
| Brooks, Celine; Snoberger, Aaron; Belcastro, Marycharmain et al. (2018) Archaeal Unfoldase Counteracts Protein Misfolding Retinopathy in Mice. J Neurosci 38:7248-7254 |
| Kim, Young-Chan; Snoberger, Aaron; Schupp, Jane et al. (2015) ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function. Nat Commun 6:8520 |
